Fluorescence imaging is found at the heart of numerous chemical and biomedical analysis schemes. Many of these schemes are based on the introduction of a fluorescent species as a marker, stain, dye or indicator [J-M. Devoisselle et al., Optical Engineering 32(2), 239 (1993); R. P. Haugland and A. Minta, “Design and Application of Indicator Dyes,” Noninvasive Techniques in Cell Biol., ed. B. H. Satir, Chap. 1, p 1, (Wiley-Liss, New York, N.Y., 1990); D. J. Gross, “Quantitative Single Cell Fluorescence Imaging of Indicator Dyes,” Noninvasive Techniques in Cell Biol., ed. B. H. Satir, Chap. 2, p 21, (Wiley-Liss, New York, N.Y., 1990)].
Organic chelates derived from lanthanide ions have become increasingly important as sensitive fluorescent markers for time resolved fluorometric assays [E. P. Diamandis, Clin. Biochem. 21, 139-150 (1988); Clin. Chim. Acta. 194, 19-50 (1990); Anal. Chem. 62, 11 49A-11 57A (1990); E. Soini and T. Lovgren, Crit. Rev. Anal. Chem. 18, 105-154 (1987)]. In particular, terbium and europium complexes are of significant value for these applications because of the efficient fluorescent emission in the visible region (E. P. Diamandis, U.S. Pat. No. 5,312,922). Both of these ions display a weak fluorescent emission in their non-complexed form, but when chelated with an appropriate organic ligand this visible emission is dramatically enhanced. Thus, the organic ligand acts as an antenna for absorbing ultraviolet radiation and transferring this energy to the metal ion that then dissipates the absorbed energy in the form of visible light. The mechanistic details of this phenomenon are well studied and have been extensively documented [A. P. B. Sinha, Fluorescence and Laser Action in Rare Earth Chelates/Spectroscopy in Inorganic Chemistry Volume II, Academic Press, (1971)].
There are numerous chelates capable of long-lived fluorescence but not all of these complexes are suitable for biological applications, one reason being due to their instability in aqueous media [G. Kallistratos, Fluorescent Properties of Aromatic Complexes with Rare Earths and Other Elements of the IIIa-Group/Chemika Chronika. New Series, 11, 249-266 (1982)]. In fact, a large majority of fluorescent chelates are operative in non-aqueous conditions only. This is largely due to the instability of the complex in aqueous solutions resulting in non-complexed metal being present and quenching of the fluorescent pathway responsible for visible light emission. Ultimately, complexes of this type would not be sensitive markers at low concentrations and would present toxicity problems in vivo because of metal deposition in soft tissue.
In recent years chelating agents based upon tetraazamacrocyclic backbones have proven to be extremely valuable for generating aqueous stable lanthanide chelates. In particular, aminocarboxylate and aminophosphonate chelating agents derived from 1,4,7,10-tetraazacyclododecane have been shown to form highly stable lanthanide chelates [W. P. Cacheris, A. D. Sherry, Inorg. Chem. 26, 958-960 (1987); J. Simón, J. R. Garlich, D. A. Wilson, and K. McMillan, U.S. Pat. No. 4,976,950]. The superior nature of this class of chelates has made them useful for diagnostic and therapeutic medical applications such as magnetic resonance imaging and bone marrow ablation. In addition, certain types of these macrocyclic chelating agents incorporating an aromatic moiety, such as the pyridine nucleus, have displayed very efficient fluorescent properties with terbium and europium (J. Kankare, J. Takalo, and P. Pasanen, U.S. Pat. No. 4,920,195). In this patent Kankare et al. demonstrate that a 14-menber macrocyclic europium chelate containing a pyridine nucleus can be conjugated to human IgG. The resulting conjugate thus contains a highly sensitive fluorescent tag (the chelate) which can be quantified by fluorescent immunoassay procedures.
Use of paramagnetic macrocyclic chelates based upon gadolinium (Gd) as contrast agents for magnetic resonance imaging has attracted considerable attention. The appeal of the lanthanide chelates is directly attributed to their kinetic and thermodynamic stability under the challenging aqueous environment encountered in the human body. Appropriate modifications can be made to this type of ligand that will cause pronounced fluorescence when lanthanides, such as terbium (Tb) and europium (Eu), are at the central core. Kim et al., Inorg. Chem. 34, 2233-43 (1995), have reported a recent study on some potential MRI contrast agents based upon macrocyclic pyridine containing ligands. In this study, the inner sphere water coordination was determined by measuring the fluorescent properties of the terbium and europium chelates.
The importance of macrocyclic lanthanide chelates for medical applications has continued to grow with the development of tissue specific agents. Thus far, applications have focused on chelation of radioactive and paramagnetic metal ions for therapy and diagnosis (J. Simón, J. R. Garlich, D. A. Wilson, K. McMillan, U.S. Pat. No. 4,976,950; examples of gadolinium chelates for MRI are Prohance™ by Squibb and Dotarem™ by Guerbet). However, these chelates do not have any fluorescent properties.
The use of fluorescent chelates as visual tissue specific agents was discussed in U.S. Pat. No. 5,928,627 (G. E. Kiefer and D. J. Bornhop). These are macrocyclic lanthanide chelates that fluoresce when excited with UV light in the relatively short 260-280 nm wavelength range that can be used to detect colon cancer visually when illuminated with UV light.
Each year about 31,000 Americans develop oral cancer (4% of all cancers in males and 2% in females). [E. Baden, CA Cancer J. Clin. 37(1), 49-62(1987).] About half of those cancer persons are dead within 5 years from diagnosis, and of the survivors, many will have disfiguration and/or functional compromise. Thus early diagnosis is important as this could increase survival from 50% to 80% [S. Silverman, Jr. and M. Gorsky, J. Am. Dent. Assoc. 120(5), 495-499 (1990).] The incidence of second primary carconomas in the esophagus and upper aerodigestive tract has been estimated to be between 2-30% [D. P. Varbec, Trans. Pa. Acad. Ophthalmol. Otolaryngol. 32(2), 177-191 (1979); P. H. Marks and F. G. Schechter, Ann. Thorac. Surg. 33(4), 324-332 (1982); J. Gluckman, Laryngoscope 3, 90 (1983)]. Detection of these second primary tumors in the early stages would be advantageous.
The most widely used non-invasive method now used for early detection of oral cancer is gross visualization under white-light illumination. This method can be problematic due to the low visual contrast for abnormal tissue, particularly for early detection of dysplastic and pre-malignant lesions where discrimination of such lesions from non-malignant lesions is very difficult. Spectroscopic techniques have been developed to try to improve early detection. Contrast agents are used to enhance the spectroscopic contrast between normal and diseased tissue. Most contrast agents require systemic administration to be effective, which causes exposure to phototoxicity for the tissues. A non-invasive administration of these contrast agents would be preferred.
Thus far, commercial applications of fluorescent chelates have been restricted primarily to the labeling of proteins and antibodies for immunoassays [E. P. Diamandis, Clinica Chimica Acta 194, 19-50 (1990); U.S. Pat. No. 5,312,922]. Products such as FIAgen™ (CyberFluor Inc., Toronto, Ontario, Canada) are available and utilize the europium chelate of 4,7-bis(chlorosulfonyl)-1,10-phenanthroline-2,9-dicarboxylic acid as the fluorescent label. Fluorescent labels of this type are extremely sensitive and can be detected in the subpicomolar range using time resolved fluorometry.
One of the most important features of diagnostic agents is that they must enhance the accuracy of assessing a disease state. Most frequently this involves delivering the diagnostic agent to a specific organ or soft tissue where a suspected abnormality may be present. Currently, the covalent attachment of a small molecule (i.e., diagnostic fragment) to a large protein or antibody (referred to as “bifunctional”) is receiving much attention as the method of choice for achieving tissue specificity.
One such example of a bifunctional molecule is disclosed in Griffin, J. M. M. et al, “Simple, high yielding synthesis of trifunctional fluorescent lanthanide chelates”, Tetrahedron Letters 42 (2001) pp. 1-3. Griffin discloses a lanthanide chelating ligand based on the cyclen (1,4,7,10-tetraazacyclododecane) nucleus which possesses a single carboxyl group for conjugation to a biologically active species such as an antibody. However, this method is inherently complex and expensive since it requires the use of a specialized antibody in order to achieve tissue specificity.
Therefore, it would be advantageous to use a small molecule diagnostic agent that would localize in a specific tissue of the body without the need for attachment to a delivery molecule such as an antibody. Furthermore, if a stable, fluorescent lanthanide chelate were to exhibit tissue specificity, it would be possible to visually determine the presence of the chelate by illuminating with the appropriate light source that minimized soft tissue damage. Potential applications would be fluorescent guided surgical procedures, in vivo imaging of bone or soft tissue cell growth or morphology, and examinations of the gastrointestinal tract.